S100 proteins constitute a major subfamily of EF-hand Ca2+-binding proteins that are characterized by cell type-specific expression and unusually high abundance in a variety of disease states, including arthritis, cancer, cystic fibrosis and AIDS. These proteins are distinguished from other EF-hand proteins by their unique N-terminal Ca2+ binding sites and high affinity for Zn2+. The S100s appear to take part in Ca2+ signalling pathways that are distinct from those controlled by the prototypical EF-hand Ca2+ sensors (eg. calmodulin and troponin C), but also have proposed functions other than in Ca2+ signalling. A considerable amount of data has been accumulated on the structure and other biophysical properties of S100 proteins. However, there is a critical gap in knowledge because nearly all of these studies have been carried out on either apo and/or Ca2+-loaded states in the absence of cellular targets. The research proposed in this application is focused on the most important outstanding question regarding S100 protein structural biology: What are the effects of binding to cellular targets on S100 protein structure, dynamics and ion affinity? A multi-disciplinary strategy that incorporates biochemical and structural approaches will be utilized to address these critical questions. The collaboration between the Chazin and Kuznicki groups couples biophysical and structural research to those of biologists in Poland who are also working to elucidate S100 protein function, to help ensure the biological significance of the structural results. The broad, long-term objectives of this research program are to understand the structural basis for the distinct cellular activities of S100 proteins, so that we may address their roles in health and disease. In this proposal, we will determine the structure of the complex of S100A6 with its target calcyclin binding protein. The Polish team will clone, produce and characterize the protein. The US team will carry out solution NMR experiments and structure calculations and analysis. This research will be done primarily in Poland as an extension of NIH grant # R01 GM62112.